Mechanistic Insight with HBCH(2)CoA as a Probe to Polyhydroxybutyrate (PHB) Synthases

[Image: see text] Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1–2 MDa. A substrate analogue HBCH(2)CoA, in which the S in HBCoA is replaced with a CH(2) group, was synthesized in 13 steps using a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor of a class I and a class III PHB synthases, with K(is) of 40 and 14 μM, respectively. To probe the elongation steps of the polymerization, HBCH(2)CoA was incubated with a synthase acylated with a [(3)H]-saturated trimer-CoA ([(3)H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [(3)H]-sTCoA ([(3)H]-sT-CH(2)-CoA), saturated dimer-([(3)H]-sD-CO(2)H), and trimer-acid ([(3)H]-sT-CO(2)H), distinct from the expected methylene analogue of [(3)H]-saturated tetramer-CoA ([(3)H]-sTet-CH(2)-CoA). Detection of [(3)H]-sT-CH(2)-CoA and its slow rate of formation suggest that HBCH(2)CoA may be reporting on the termination and repriming process of the synthases, rather than elongation.